It is desirable to swiftly and simply, reliably identify microbes. Reliable identification allows the pathogenicity and/or other characteristics of a microbial sample to be identified. Determining the biomolecule make-up of a microbe can assist in identification and hence diagnosis.
Mass spectrometry can be used to analyse biomolecules, typically using soft ionization techniques. Matrix-Assisted Laser Desorption/lonization (MALDI)-MS is one such technique, and has been widely used in the analysis of large biomolecules including proteins. Protein fingerprinting has enabled the application of MALDI-MS for microbial identification and diagnostics.
However, protein fingerprinting cannot reliably differentiate between different strains of the same species. Serological testing or PCR analysis is needed to test the pathogenicity of a sample.
Lipids are known to be structurally important within cells, and are the major components of cell membranes and other sub-cellular structures. They are active in major cellular mechanisms, and influence the properties and functionality of proteins.
US 2012/0197535 describes glycolipid (saccharolipid) extraction from bacterial samples. Analysis of the glycolipid make-up using mass spectrometry techniques (including MALDI) allows identification of bacterial strains. This technique exploits the specificity of bacterial outer cell wall glycolipid structures (e.g. Lipid A, lipoteichoic acid). However, this method was developed for and tailored to the specific extraction of these glycolipids and the inventors have found that it is not widely applicable. For example, it cannot be used to determine the identity of other microbial organisms (e.g. yeasts or fungi), which do not contain these glycolipids.
The cellular structure of microbes can vary greatly between different genera (e.g. bacteria, yeast, filamentous fungi), and even within a genus at the species level. For example, prokaryotic organisms (e.g. bacteria) are divided into two major groups, Gram+ and Gram−, depending on the characteristics of their cell structures. However, despite the quite different architecture of the cell structures in prokaryotes and eukaryotes (e.g. yeasts and fungi), all have a cytoplasmic membrane.
Phospholipids (PLs) (particularly glycerophospholipids and phosphosphingolipids) are known to be the major membrane lipids of many microbes. Indeed, components of phospholipid biosynthesis and the PLs themselves are membrane bound and serve as the precursors of other cell membrane components.[1]
For example, cationic phosphatidylcholine (PC) accounts for about 50% of total membrane lipids in eukaryotes alongside phosphatidylserine (PS) and phosphatidylinositol (PI) as minor components. Zwitterionic phosphatidylethanolamine (PE), anionic phosphatidylglycerol (PG) and/or cardiolipin (CL) represent the major membrane lipids of most known bacteria (e.g. up to 80% PE in E. coli).[2] Other significant PLs are amino acid esters of PG (e.g. lysyl -, alanyl-,or ornithyl-PG)[14].
Some other neutral lipids (NLs) (or non-polar lipids), such as diacylglycerols (DAGs), triacylglycerols (TAGs) and cholesteryl esters (CEs), are known to also play an important role in cell membrane functionality. Although CEs are only rarely found in prokaryotes (i.e. bacteria) they are more common lipid components of eukaryotes (i.e. yeasts, fungi and mammalian cells). These lipid molecules contain 1-3 fatty acids esterified to a glycerol or sterol backbone. A significant group of NLs is the glycosyl diglycerides (e.g. digalactosyldiglyceride, DGDG), which are found mainly in Gram+ bacteria (e.g. Bacillus spp.)[14].
The importance of PLs (and NLs) for cell membranes, means that homeostasis of the lipid composition is very important to sustain the cell membrane integrity and functionality (e.g. trans-membrane signalling, intercellular interactions, energy metabolism, cell proliferation, etc.) of microbial cells and their viability in different environments.[3] It is well known that alterations to cellular PL compositions can result from exposure of microbes to environmental stresses, such as extreme temperatures, toxic substances, food additives and antibiotics.[4][5][6][7]
Consequently, microbial cells possess a very characteristic, evolutionary based phospholipid composition that can be used for chemotaxonomic purposes, whereby small differences in the phospholipid profiles can potentially be exploited for differentiation at the strain level. This allows for strains with certain characteristics to be identified, including those that are pathogenic and resistant to antibiotics.
Microbial phospholipid compositions are very complex, and are typically analysed using mass spectrometry (MS) because it allows the simultaneous detection of many individual molecular species within a single MS spectrum. One of the first examples of using MS for the classification of microorganisms was the use of gas-chromatography (GC)-MS.[8] GC-MS is used for cellular fatty acid analysis because of its good chromatographic separation potential.[9] However, lipids need to be broken down into constituent fatty acid molecules to be analysed, and thus the phospholipid composition within the cells cannot be determined.
Functional analysis of phospholipids suggests that the structural properties (e.g. head group, chain length, degree of unsaturation) are primarily responsible for membrane function.[10] This information is also lost when phospholipids are broken down into their constituent fatty acids.
Accordingly, detection techniques conserving intact phospholipid structures during analysis are more appropriate for chemotaxonomical purposes. Some reports have shown the usefulness of intact lipid profiling for differentiation of bacteria using softer-ionisation techniques like fast-atom bombardment (FAB)[11] and electrospray ionization (ESI)-MS.[12]
However, MALDI-MS is generally a preferred soft-ionisation technique. Data can be obtained simply and quickly, and is relatively simple to analyse because the molecules are almost exclusively detected as singly charged ions upon laser irradiation of the matrix-embedded samples. Further the instruments used are robust and reliable allowing the analysis of crude (i.e. unpurified) samples. Consequently, MALDI-MS has developed into a routine technique for microbial diagnostics based on “protein fingerprinting”.[13] 
Recently, MALDI-MS has been reported as a method for bacterial phospholipid analysis.[14][15] However, a lack of reliable sample preparation protocols and instrumentation techniques has prevented wider application of these methods. Routine bacterial identification by phospholipid analysis using MALDI-MS has not been possible for closely related bacteria, preventing its use in taxonomy. Moreover, no successful lipid MALDI-MS analysis has been reported for other types of microbe (e.g. yeasts and fungi), which must still be identified by cell morphology using light microscopy, genotyping, and/or protein fingerprinting.
Furthermore, differentiation of microbial strains (within a species) using MALDI-MS based phospholipid profiling has not been reported. Nor has MALDI-MS based lipid profiling of yeasts or fungi.